Organ protection in PSMA-targeted radionuclide therapy of prostate cancer

ABSTRACT

A method of reducing radiation exposure of a non-cancerous tissue of a patient diagnosed with a cancer includes administering to the patient an agent capable of competing for binding sites on a surface of the non-cancerous tissue, provided that the administration is carried out after a waiting period that follows administration of a compound including a radionuclide to the patient, the compound having affinity for both a cancerous tissue and the non-cancerous tissue, and, further provided that the binding sites have an affinity for both the agent and the compound.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/934,304, filed on Mar. 23, 2018, which is a continuation of U.S.patent application Ser. No. 14/846,252, filed on Sep. 4, 2015, now U.S.Pat. No. 9,956,305, which claims priority from U.S. Provisional PatentApplication No. 62/047,546, filed on Sep. 8, 2014. The contents of theseapplications are incorporated herein by reference in their entiretiesfor any and all purposes.

FIELD

The present technology is generally related to radionuclide therapy.More particularly, it is related to preventing or minimizing ancillaryradiation damage of non-target, normal tissues by radionuclides duringradionuclide therapy for cancerous tissue.

BACKGROUND

The cell surface peptidase, prostate-specific membrane antigen (PSMA)demonstrates intense overexpression in the majority of both primary andmetastatic prostate cancers (PCa) and is expressed on the neovasculatureof the majority of solid tumors including bladder, colon, liver, lung,renal, glioblastoma multiforme and melanoma (28). This along with apositive correlation of PSMA with traditional adverse prognostic factorsaroused growing interest in evaluation of this target.¹⁻³ Initially,antibodies (mAB) targeting PSMA have been used for PCa imaging andtherapeutic approaches. The ¹¹¹In labeled mAB capromab pendetide(ProstaScint, EUSA Pharma, Langhorne, Pa.) was approved in the USA in1996. However, because the mAB, 7E11 targeted the intracellular domainof PSMA, its diagnostic value was rather limited.⁴ Another monoclonalPSMA antibody, J591 targets the extracellular domain of PSMA, but likemost complete mABs it presents with a slow tumor accumulation and a longcirculation time in blood. Thus, diagnostic mAB-tracers requireprolonged imaging—even days after injection.⁵ Transferred toradionuclide therapy mABs commonly translate into an unfavorabledosimetry with pronounced hematotoxicity.⁶ Recently, the development ofthe Glu-urea-based high affinity small molecule PSMA inhibitors MIP1072and MIP1095, either labeled with ¹²³I for imaging or ¹³¹I for targetedradionuclide therapy, rendered rapid tumor uptake possible.⁷⁻⁹ SincePSMA is internalized through clathrin-coated pits²⁷ either spontaneouslyor after binding of an antibody or an inhibitor, it is also an excellenttarget for endoradiotherapy. In this respect, the accumulation of thetracer in normal tissues has to be considered to prevent or diminish theextend of side effects, particularly in normal tissues that express lowlevels of PSMA including the proximal renal tubules of the kidney,salivary glands, and the brush-border epithelium of the smallintestines.²⁸

In a positron emission tomography (PET)-based dosimetry study of¹²⁴I-MIP1095, doses of up to 300 Gy in lymph node and bone metastases ofcastration refractory prostate cancer (CRPCa) were calculated. Theorgans with the highest off-target radiation doses were salivary glands(3.8 mSv/MBq), liver (1.7 mSv/MBq) and kidneys (1.4 mSv/MBq), while redmarrow was 0.37 mSv/MBq.¹⁰ Therefore, kidneys may be the limiting factorfor the maximum activity that can cumulatively be administered safely.However, improving kidney uptake without losing tumor dose is a realchallenge because PSMA is physiologically expressed in the kidneytubules.¹ The pharmacokinetics of MIP-1072 and MIP-1095 in animals havealready been evaluated in detail.⁷ The authors reported a similaraccumulation of both compounds in PSMA-expressing LNCaP xenografts butwith very different pharmacokinetic profiles. MIP-1072 clears rapidlyfrom target (tumor) and non-target (normal) tissues. In contrast,MIP-1095 presents a longer biological half-life in tumor but not inkidneys, which corresponds to a higher fraction of ligand inducedreceptor internalization and retention in tumor cells.

For the development of Glu-Urea based PSMA ligands, the structurallyunrelated PSMA inhibitor 2-(phosphonomethyl)pentanedioic acid (PMPA) iscommonly used as a competitor in blocking studies to demonstrate thespecific binding of the molecule of interest, PSMA. In this respectsimultaneous coinjection of 50 mg/kg PMPA resulted in a completeblocking of radiolabeled-MIP-1095 to binding sites in tumor andkidneys.⁷ The present disclosure describes a surprising discoveryinvolving selective displacement by blocking agents of radionuclidesfrom non-target, normal tissue and organs while retaining radionuclideat target, cancerous tissue sites through non-simultaneousadministration methods.

SUMMARY

Disclosed herein, in one aspect, are methods of Aing radiation exposureof a non-cancerous tissue of a patient diagnosed with a cancer, themethods comprising administering to the patient an agent capable ofcompeting for binding sites on a surface of the non-cancerous tissue,provided that the administration is carried out after a waiting periodthat follows administration of a compound comprising a radionuclide tothe patient, the compound having affinity for both a cancerous tissueand the non-cancerous tissue, and, further provided that the bindingsites have an affinity for both the agent and the compound.

In another aspect, disclosed herein are methods of reducing radiationexposure of a non-cancerous tissue of a patient diagnosed with a cancer,the methods comprising administering to the patient an effective amountof an agent, allowing a waiting period to pass and administering aneffective amount of a compound comprising a radionuclide, the agentbeing capable of competing with the compound for binding sites on asurface of the non-cancerous tissue.

In another aspect, disclosed herein are methods of radionuclide therapyfor treating prostate cancer in a subject, the methods comprisingadministering a compound comprising a radionuclide to the subject,allowing a waiting period of about 10 hours to about 36 hours followingthe administration of the compound, and then administering an agent tothe subject, wherein the agent comprises a recognition moiety forbinding sites of the compound in non-cancerous tissue, wherein thebinding sites have an affinity for both the agent and the compound.

In another aspect, disclosed herein are methods of radionuclide therapyfor treating prostate cancer in a subject, the methods comprisingadministering an agent to the subject, allowing a waiting period ofabout 10 hours to about 36 hours following the administration of theagent, and then administering a compound comprising a radionuclide tothe subject, wherein the agent comprises a recognition moiety forbinding sites of the compound in non-cancerous tissue, wherein thebinding sites have an affinity for both the agent and the compound.

In another aspect, disclosed herein are methods of reducing ancillaryradiation damage to non-cancerous tissue during radiotherapy for cancertreatment in a subject harboring cancerous tissue, the methodscomprising administering a compound comprising a radionuclide to thesubject, allowing a waiting period, and administering an agent to thesubject after completion of the waiting period, wherein the agent iscapable of reducing a concentration of the compound in the non-canceroustissue to a greater extent than a concentration of the compound in thecancerous tissue.

In another aspect, disclosed herein are methods of reducing ancillaryradiation damage to non-cancerous tissue during radiotherapy for cancertreatment in a subject harboring cancerous tissue, the methodscomprising administering an agent to the subject, allowing a waitingperiod, and administering a compound comprising a radionuclide to thesubject after completion of the waiting period, wherein the agent iscapable of reducing a concentration of the compound in the non-canceroustissue to a greater extent than a concentration of the compound in thecancerous tissue.

In another aspect, disclosed herein are therapeutic radionuclideregimens for treating cancerous tissue in a subject, the regimenscomprising administering a compound comprising a radionuclide to asubject harboring a cancerous tissue, and administering an agent to thesubject after allowing a waiting period to pass, in which the agent isconfigured to reduce radionuclide concentration in non-cancerous tissuerelative to a concentration of radionuclide in the non-cancerous tissueprior to administration of the agent.

In another aspect, disclosed herein are therapeutic radionuclideregimens for treating cancerous tissue in a subject, the regimenscomprising administering an agent to a subject harboring a canceroustissue, and administering a compound comprising a radionuclide to thesubject after allowing a waiting period to pass, in which the agent isconfigured to reduce radionuclide concentration in non-cancerous tissuerelative to a concentration of radionuclide in the cancerous tissue.

In another aspect, disclosed herein are therapeutic radionuclideregimens for treating cancerous tissue in a subject, the regimenscomprising administering a compound comprising a radionuclide to asubject harboring a cancerous tissue, and administering an agent to thesubject after allowing a waiting period to pass, in which the agent isconfigured to compete with the compound for binding sites on a surfaceof non-cancerous tissue.

In another aspect, disclosed herein are therapeutic radionuclideregimens for treating cancerous tissue in a subject, the regimenscomprising administering an agent to a subject harboring a canceroustissue, and administering a compound comprising a radionuclide to thesubject after allowing a waiting period to pass, in which the agent isconfigured to compete with the compound for binding sites on a surfaceof non-cancerous tissue.

In another aspect, disclosed herein are treatment protocols for asubject diagnosed with cancer, the protocols comprising: administering acompound comprising a radionuclide to the subject, allowing a waitingperiod to pass, successively administering to the subject multiple,low-concentration doses of an agent capable of reducing a radionuclideconcentration in non-cancerous tissue in the subject to a greater extentthan a radionuclide concentration in cancerous tissue in the subject,observing between successive doses of the agent a concentration changeof the compound in non-cancerous tissue until a desired non-canceroustissue concentration of the radionuclide is obtained, and discontinuingadministration of the agent.

In another aspect, disclosed herein are kits comprising:

a) a compound comprising a radionuclide;

b) a blocking agent for reducing ancillary radiation damage tonon-cancerous tissue, wherein both the compound and the agent comprise arecognition moiety for prostate specific membrane antigen.

In another aspect, disclosed herein are treatment protocols for asubject diagnosed with cancer, the protocols comprising:

a) administering to the subject a compound comprising a radionuclide,

b) allowing a waiting period to pass,

c) administering to the subject an agent in an amount sufficient tocause a displacement of radionuclide in non-cancerous tissue andretention of radionuclide in cancerous tissue.

In another aspect, disclosed herein are treatment protocols for asubject diagnosed with cancer, the protocols comprising:

a) administering to the subject an agent,

b) allowing a waiting period to pass,

c) administering to the subject a compound comprising a radionuclide,wherein the compound is administered in an amount sufficient to cause adisplacement of the agent in cancerous tissue.

In another aspect, disclosed herein are methods of reducing exposure ofone's non-cancerous tissue to radiation, the methods comprisingreceiving an agent comprising a recognition moiety, wherein therecognition moiety is for a binding site of a compound, and the compoundcomprises a radionuclide, provided that the receiving step is carriedout after a waiting period that follows a prior step of receiving thecompound comprising the radionuclide.

In another aspect, disclosed herein are methods of reducing exposure ofone's non-cancerous tissue to radiation, the methods comprisingreceiving a compound comprising a radionuclide, provided that thereceiving step is carried out after a waiting period that follows aprior step of receiving an agent comprising a recognition moiety,wherein the recognition moiety is for a binding site of the compound.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a chart illustrating the experimental setup with the firstimage at 16 hours post-injection of the compound comprising aradionuclide, [¹²⁵I] MIP-1095. Immediately after scintigraphy, saline(control group) or different doses of the blocking agent, PMPA, weregiven and additional imaging was conducted at the indicated time periodspost injection. FIG. 1B shows scintigraphic images of controls and PMPAtreated animals, according to the examples.

FIG. 2A is a time course of residual radioactivity in the kidney, andFIG. 2B is a time course of residual radioactivity in the tumorexpressed in % of the value at 16 hours post injection of [¹²⁵I]MIP-1095 using different doses of PMPA, according to the examples.

FIG. 3A is a chart showing the experimental setup with the first imageat 1 hour post-injection of the Compound comprising a radionuclide,^(99m)Tc MIP-1404. Immediately after scintigraphy saline (control group)or 50 mg/kg PMPA were administered, additional imaging was conducted atthe indicated time periods post injection. FIG. 3B shows scintigraphicimages of a control and a PMPA treated animal, according to theexamples.

FIG. 4A is a time course of residual radioactivity in the kidney, andFIG. 4B is a time course of residual radioactivity in the tumorexpressed in % of the value at 1 hour post-injection ^(99m)Tc MIP-1404,according to the examples.

FIG. 5 is a chart showing ex vivo gamma counting values by tissue (%ID/g).

FIG. 6 is a chart showing ex vivo gamma counting values by tissue (%ID/g).

DETAILED DESCRIPTION

Various embodiments are described hereinafter. It should be noted thatthe specific embodiments are not intended as an exhaustive descriptionor as a limitation to the broader aspects discussed herein. One aspectdescribed in conjunction with a particular embodiment is not necessarilylimited to that embodiment and can be practiced with any otherembodiment(s).

As used herein, “about” will be understood by persons of ordinary skillin the art and will vary to some extent depending upon the context inwhich it is used. If there are uses of the term which are not clear topersons of ordinary skill in the art, given the context in which it isused, “about” will mean up to plus or minus 10% of the particular term.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the elements (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. Recitation of ranges of values herein are merely intended toserve as a shorthand method of referring individually to each separatevalue falling within the range, unless otherwise indicated herein, andeach separate value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g., “such as”) provided herein, isintended merely to better illuminate the embodiments and does not pose alimitation on the scope of the claims unless otherwise stated. Nolanguage in the specification should be construed as indicating anynon-claimed element as essential.

It has been found that during radionuclide therapy for cancerous tissuein a subject, ancillary radiation damage of normal tissue in a subjectmay be reduced by administration of a blocking agent to the subject. Insome embodiments, the blocking agent is administered to the subjectafter an induction period for an administered radionuclide, i.e., a timeperiod during which an administered radionuclide-containing compound isallowed to localize in various tissues. In other embodiments, theblocking agent is administered to the subject prior to theadministration of a radionuclide. The radionuclide-containing compoundsdescribed herein have a recognition moiety for a cancerous (i.e., tumor)tissue For example, in some embodiments, the compound may have arecognition moiety for prostate specific membrane antigen (PSMA), andthe compound may contain a radionuclide as part of the compound, or thecompound may have a chelation group bound to a radionuclide. In oneembodiment, the compound is a Glu-Urea-based PSMA ligand. The blockingagents are materials which are not radiolabeled and will displace theradionuclide-containing compound from normal (i.e., non-cancerous ornon-tumor) tissue, while not have any effect, or at least a lessereffect on displacement of the radionuclide-containing compound from thecancerous tissue. For example, in some embodiments, the blocking agenthas a recognition moiety for prostate specific membrane antigen. Infurther embodiments, the binding sites on a surface tissue have anaffinity for both the compound and the blocking agent. In someembodiments, the blocking agent is a compound without a radiolabel or acold compound.

In one aspect, a method is provided for reducing radiation exposure of atissue of a patient diagnosed with a cancer, which tissue issubstantially free of tumor tissue. In some embodiments, the methodincludes administering to the patient an agent capable of competing forbinding sites on a surface of the tissue, provided that theadministration is carried out after a waiting period that followsadministration of compound having a radionuclide to the patient, and,further provided that the binding sites have an affinity for both theagent and the radionuclide. In other embodiments, the method includesadministering to the patient a compound having a radionuclide, providedthat the administration is carried out after a waiting period thatfollows administration of an agent to the patient, the agent beingcapable of competing for binding sites on a surface of the non-canceroustissue, and the compound having affinity for both a cancerous tissue andthe non-cancerous tissue, and, further provided that the binding siteshave an affinity for both the agent and the compound.

In some embodiments disclosed herein are methods of reducing radiationexposure of a non-cancerous tissue of a patient diagnosed with a cancer,the method including administering to the patient an agent capable ofcompeting for binding sites on a surface of the non-cancerous tissue,provided that the administration is carried out after a waiting periodthat follows administration of a compound comprising a radionuclide tothe patient, the compound having affinity for both a cancerous tissueand the non-cancerous tissue, and, further provided that the bindingsites have an affinity for both the agent and the compound. In someembodiments, the agent displaces bound radionuclide from the bindingsites thereby reducing the exposure of the non-cancerous tissue. In someembodiments, the agent does not comprise a radionuclide. In someembodiments, the agent comprises the compound without the radionuclide.

In some embodiments disclosed herein are methods of reducing radiationexposure of a non-cancerous tissue of a patient diagnosed with a cancer,the method including administering to the patient an effective amount ofa compound comprising a radionuclide, allowing a waiting period to pass,and administering an effective amount of an agent capable of competingwith the compound for binding sites on a surface of the non-canceroustissue. In some embodiments, the compound exhibits an affinity for botha cancerous tissue and the non-cancerous tissue. In some embodiments,the binding sites have an affinity for both the agent and the compound.In some embodiments, the agent displaces bound radionuclide from thebinding sites thereby reducing the exposure of the non-cancerous tissue.In some embodiments, the agent does not comprise a radionuclide. In someembodiments, the agent comprises the compound without the radionuclide.

In some embodiments disclosed herein are methods of reducing radiationexposure of a non-cancerous tissue of a patient diagnosed with a cancer,the methods comprising administering to the patient an effective amountof an agent, allowing a waiting period to pass and administering aneffective amount of a compound comprising a radionuclide, the agentbeing capable of competing with the compound for binding sites on asurface of the non-cancerous tissue. In some embodiments, the compoundexhibits an affinity for both a cancerous tissue and the non-canceroustissue. In some embodiments, the binding sites have an affinity for boththe agent and the compound. In some embodiments, the agent does notcomprise a radionuclide. In some embodiments, the agent comprises thecompound without the radionuclide.

In another aspect, a method of radionuclide therapy is provided fortreating cancerous tissue in a subject. The method includesadministering a compound having a radionuclide, i.e., aradionuclide-containing compound, to the subject harboring the canceroustissue, allowing a waiting period to pass, and administering an agent tothe subject. The agent is configured to reduce radionuclideconcentration in normal or non-cancerous tissue. In some embodiments,the cancerous tissue is prostate cancer or metastatic prostate cancer.The non-cancerous tissue may be wide range of normal bodily tissues,including, but not limited to kidney tissue, salivary gland tissue,lacrimal gland tissue, parotid gland tissue, and small intestine tissue.

In some embodiments disclosed herein are methods of radionuclide therapyfor treating prostate cancer in a subject, the method comprisingadministering a compound comprising a radionuclide to the subject,allowing a waiting period of about 10 hours to about 36 hours followingthe administration of the compound, and then administering an agent tothe subject, wherein the agent comprises a recognition moiety forbinding sites of the compound in non-cancerous tissue, wherein thebinding sites have an affinity for both the agent and the compound.

In another aspect, a method of radionuclide therapy is provided fortreating cancerous tissue in a subject. The method includesadministering an agent to the subject harboring the cancerous tissue,allowing a waiting period to pass, and administering to the subject acompound having a radionuclide, i.e., a radionuclide-containingcompound. The agent is configured to reduce radionuclide concentrationin normal or non-cancerous tissue. In some embodiments, the canceroustissue is prostate cancer or metastatic prostate cancer. Thenon-cancerous tissue may be wide range of normal bodily tissues,including, but not limited to kidney tissue, salivary gland tissue,lacrimal gland tissue, parotid gland tissue, and small intestine tissue.

In some embodiments disclosed herein are methods of radionuclide therapyfor treating prostate cancer in a subject, the methods comprisingadministering an agent to the subject, allowing a waiting period ofabout 10 hours to about 36 hours following the administration of theagent, and then administering a compound comprising a radionuclide tothe subject, wherein the agent comprises a recognition moiety forbinding sites of the compound in non-cancerous tissue, wherein thebinding sites have an affinity for both the agent and the compound.

In another aspect, a method of reducing ancillary radiation organ damageduring radiotherapy for cancer treatment of a subject is provided. Insome embodiments, the method includes administering a compound includinga radionuclide, as defined above, to the subject, allowing an inductionperiod, and administering an agent to the subject after completion ofthe induction period. In other embodiments, the method includesadministering an agent to the subject, allowing an induction period, andadministering to the subject a compound including a radionuclide, asdefined above, after completion of the induction period. The agent isconfigured to reduce radionuclide concentration in non-cancerous tissue.

In another aspect, a method of reducing ancillary radiation damage tonon-cancerous tissue during radiotherapy for cancer treatment in asubject harboring cancerous tissue is provided. In some embodiments, themethod includes administering a compound comprising a radionuclide tothe subject, allowing a waiting period, and administering an agent tothe subject after completion of the waiting period, wherein the agent iscapable of reducing a radionuclide concentration in the non-canceroustissue to a greater extent that a radionuclide concentration in thecancerous tissue. In other embodiments, the method includesadministering an agent to the subject, allowing a waiting period, andadministering to the subject a compound comprising a radionuclide aftercompletion of the waiting period, wherein the agent is capable ofreducing a radionuclide concentration in the non-cancerous tissue to agreater extent that a radionuclide concentration in the canceroustissue.

In another aspect, a treatment protocol for a subject diagnosed withcancer is provided. The protocol includes administering a compoundhaving a radionuclide to the subject, allowing a waiting period to pass,successively administering to the subject multiple, low-concentrationdoses of an agent capable of reducing a radionuclide concentration innon-cancerous tissue in the subject to a greater extent that aradionuclide concentration in cancerous tissue in the subject, observingbetween successive doses of the agent a concentration change of thecompound in non-cancerous tissue until a desired non-cancerous tissueconcentration of the radionuclide is obtained, and discontinuingadministration of the agent. Accordingly, the treatment protocol isessentially a titration of the compound from the body of the subject.Low-concentration doses are used such that large amounts of the compoundare not displaced from tissue to which it is bound as a result of anysingle administered dose. Rather, the displacement of the compound fromnon-cancerous tissue is approached slowly such that higher radionuclideconcentrations may be maintained in target, or cancerous tissue. As usedherein, a low concentration dose concentration may be from about 0.01mg/kg to about 10 mg/kg. In other embodiments, the low concentrationdose is from about 0.2 mg/kg to about 10 mg/kg. In some embodiments, thelow concentration dose is from about 0.01 mg/kg to about 5 mg/kg. Insome embodiments, the low concentration dose is from about 0.01 mg/kg toabout 1 mg/kg. In some embodiments, the low concentration dose is fromabout 0.01 mg/kg to about 0.5 mg/kg. In some embodiments, the lowconcentration dose is from about 0.01 mg/kg to about 0.1 mg/kg. In someembodiments, the low concentration dose is from about 0.2 mg/kg to about5 mg/kg.

In another aspect, a method of radionuclide therapy for treatingprostate cancer in a subject is provided. The method includesadministering a compound including a radionuclide, as defined above, tothe subject, allowing a waiting period of about 10 hours to about 36hours, and administering a binding agent to the subject. In someembodiments, the binding agent is selected from cold MIP-1555, coldMIP-1519, cold MIP-1545, cold MIP-1427, cold MIP-1428, cold MIP-1379,cold MIP-1072, cold MIP-1095, cold MIP-1558, cold MIP-1405, and coldMIP-1404.

In another aspect, a method of radionuclide therapy for treatingprostate cancer in a subject is provided. In some embodiments, themethod includes administering a compound including a radionuclide, asdefined above, to the subject, allowing a waiting period of about 10hours to about 36 hours, and administering a binding agent to thesubject. In other embodiments, the method includes administering abinding agent to the subject, allowing a waiting period of about 10hours to about 36 hours, and administering to the subject a compoundincluding a radionuclide, as defined above. In some embodiments, thebinding agent is selected from cold MIP-1555, cold MIP-1519, coldMIP-1545, cold MIP-1427, cold MIP-1428, cold MIP-1379, cold MIP-1072,cold MIP-1095, cold MIP-1558, cold MIP-1405, and cold MIP-1404.

In another aspect, a method of radionuclide therapy for treatingprostate cancer in a subject is provided. In some embodiments, themethod includes administering a compound including a radionuclide, asdefined above, to the subject, allowing a waiting period of about 10hours to about 36 hours, and administering2-(phosphonomethyl)pentanedioic acid or 2-(3-mercaptopropyl)pentanedioicacid (2-MPPA) to the subject. In other embodiments, the method includesadministering 2-(phosphonomethyl)pentanedioic acid or2-(3-mercaptopropyl)pentanedioic acid (2-MPPA) to the subject, allowinga waiting period of about 10 hours to about 36 hours, and administeringa compound including a radionuclide, as defined above, to the subject.

In another aspect, a method of radionuclide therapy for treatingprostate cancer in a subject is provided. In some embodiments, themethod includes administering a compound having a radionuclide to thesubject, allowing a waiting period of about 10 hours to about 36 hoursfollowing the administration of the compound, and then administering2-(phosphonomethyl)pentanedioic acid or 2-MPPA to the subject. In otherembodiments, the method includes administering2-(phosphonomethyl)pentanedioic acid or 2-MPPA to the subject, allowinga waiting period of about 10 hours to about 36 hours following theadministration of the compound, and then administering a compound havinga radionuclide to the subject.

In another aspect, a treatment protocol for a subject diagnosed withcancer is provided. The protocol includes (a) administering to thesubject a compound including a radionuclide, (b) allowing a waitingperiod to pass, (c) administering to the subject an agent in an amountsufficient to cause a displacement of radionuclide in non-canceroustissue and retention of radionuclide in cancerous tissue. In someembodiments, the protocol includes administering to the subject theagent at a frequency sufficient to maintain displacement of theradionuclide from the non-cancerous tissue. In other embodiments, theprotocol includes administering additional compound, followed byadditional agent. In some embodiments, at least one of steps (a) and (c)are repeated at periodic intervals. In some embodiments, steps (a), (b),and (c) are repeated. In some embodiments, step (c) is repeated. In someembodiments, the displacement is at kidney, salivary glands, parotidglands, lacrimal glands, small intestines, or combination thereof. Insome embodiments, the protocol further includes (d) monitoring treatmentby imaging using scintigraphy, SPECT, or PET. In further embodiments,the protocol further includes determining that a significant amount ofradionuclide activity remains in the non-cancerous tissue by themonitoring, and administering additional agent.

In another aspect, a treatment protocol for a subject diagnosed withcancer is provided. The protocol includes (a) administering an agent tothe subject, (b) allowing a waiting period to pass, (c) administering tothe subject a compound including a radionuclide, wherein the compound isadministered in an amount sufficient to cause a displacement of theagent in cancerous tissue. In some embodiments, the protocol includesadministering to the subject the compound at a frequency sufficient tomaintain displacement of the agent from the cancerous tissue. In otherembodiments, the protocol includes administering additional agent,followed by additional compound. In some embodiments, at least one ofsteps (a) and (c) are repeated at periodic intervals. In someembodiments, steps (a), (b), and (c) are repeated. In some embodiments,step (c) is repeated. In some embodiments, the displacement is atkidney, salivary glands, parotid glands, lacrimal glands, smallintestines, or combination thereof. In some embodiments, the protocolfurther includes (d) monitoring treatment by imaging using scintigraphy,SPECT, or PET. In further embodiments, the protocol further includesdetermining that there is insufficient radionuclide activity in thecancerous tissue by the monitoring, and administering additionalcompound. In other embodiments, the protocol further includesdetermining that a significant amount of radionuclide activity is in thenon-cancerous tissue by the monitoring, and administering additionalagent.

In another aspect, a method of reducing exposure of one's tissue toradiation is provided, which tissue is substantially free of tumortissue. In some embodiments, the methods include receiving an agentcapable of competing for binding sites on a surface of the tissue,provided that the receiving step is carried out after a waiting periodthat follows a prior step of receiving a compound comprising aradionuclide, and further provided that the binding sites have anaffinity for both the agent and the radionuclide. In other embodiments,the methods include receiving a compound comprising a radionuclide,provided that the receiving step is carried out after a waiting periodthat follows a prior step of receiving an agent capable of competing forbinding sites on a surface of the tissue, and further provided that thebinding sites have an affinity for both the agent and the radionuclide.In some embodiments, tissue which is substantially free of tumor tissuehas less than 10% tumor tissue. In some embodiments, tissue which issubstantially free of tumor tissue has less than 7% tumor tissue. Insome embodiments, tissue which is substantially free of tumor tissue hasless than 5% tumor tissue. In some embodiments, tissue which issubstantially free of tumor tissue has less than 3% tumor tissue. Insome embodiments, tissue which is substantially free of tumor tissue hasless than 2% tumor tissue. In some embodiments, tissue which issubstantially free of tumor tissue has less than 1% tumor tissue.

In another aspect, methods of reducing exposure of one's non-canceroustissue to radiation are provided. In some embodiments, the methodsinclude receiving an agent comprising a recognition moiety, wherein therecognition moiety is for a binding site of a compound, and the compoundcomprises a radionuclide, provided that the receiving step is carriedout after a waiting period that follows a prior step of receiving thecompound comprising the radionuclide. In other embodiments, the methodsinclude receiving a compound comprising a radionuclide, provided thatthe receiving step is carried out after a waiting period that follows aprior step of receiving an agent comprising a recognition moiety,wherein the recognition moiety is for a binding site of the compound. Insome embodiments, one's non-cancerous tissue includes non-cancerouskidney tissue. In some embodiments, one's non-cancerous tissue includesnon-cancerous salivary gland tissue, parotid gland tissue, or lacrimalgland tissue.

In another aspect, a kit is provided. The kit may include a compoundhaving a radionuclide; a blocking agent for reducing ancillary radiationdamage to non-cancerous tissue, wherein both the compound and the agenthave a recognition moiety for prostate specific membrane antigen. Thekits may also include instructions for use.

In another aspect, a therapeutic radionuclide regimen for treatingcancerous tissue in a subject is provided. In some embodiments, theregimen includes administering a compound comprising a radionuclide to asubject harboring a cancerous tissue, and administering an agent to thesubject after allowing a waiting period to pass, in which the agent isconfigured to reduce radionuclide concentration in non-cancerous tissuerelative to a concentration of radionuclide in the non-cancerous tissueprior to administration of the agent. In other embodiments, the regimenincludes administering an agent to a subject harboring a canceroustissue, and administering a compound comprising a radionuclide to thesubject after allowing a waiting period to pass, in which the agent isconfigured to reduce radionuclide concentration in non-cancerous tissuerelative to a concentration of radionuclide in the cancerous tissue. Insome embodiments, the regimen includes administering a compoundcomprising a radionuclide to a subject harboring a cancerous tissue, andadministering an agent to the subject after allowing a waiting period topass, in which the agent is configured to compete with the compound forbinding sites on a surface of non-cancerous tissue. In some embodiments,the regimen includes administering an agent to a subject harboring acancerous tissue, and administering a compound comprising a radionuclideto the subject after allowing a waiting period to pass, in which theagent is configured to compete with the compound for binding sites on asurface of non-cancerous tissue.

In any of the above methods or treatment protocols or regimens or kits,and unless otherwise specified, the cancer or cancerous tissue may beprostate cancer. In any of the above methods or treatment protocols orregimens or kits, and unless otherwise specified, the cancerous tissuemay be prostate cancer or metastatic prostate cancer. In any of theabove methods or treatment protocols or regimens or kits, and unlessotherwise specified, the cancerous tissue or a neovasculature associatedwith a solid cancer expresses Prostate Specific Membrane Antigen (PSMA).In any of the above methods or treatment protocols or regimens or kits,unless otherwise specified, the non-cancerous tissue may include, but isnot limited to, kidney tissue, salivary gland tissue, lacrimal glandtissue, parotid gland tissue, or small intestines. In some embodiments,the methods or treatment protocols or regimens or kits serve to reduceor prevent radiation-induced damage to non-cancerous tissue includingfor example kidney damage or development of xerostomia.

In some embodiments of any of the above methods or treatment protocolsor regimens or kits, the binding site is PSMA.

In any of the above methods or treatment protocols or regimens or kits,and unless otherwise specified, the radionuclide may be, but is notlimited to, a radioactive isotope of Ga, I, Y, Lu, Bi, Ac, Re, In, Th,or Tc. Illustrative radionuclides include, but are not limited to,¹⁸⁶Re, ¹⁸⁸Re, ⁹⁹Tc, ⁶⁸Ga, ¹¹¹In, ⁹⁰Y, ¹⁷⁷Lu, ²¹³Bi, ²²⁵Ac, ²²⁸Th, ²²⁹Th,^(229m)Th, ²³⁰Th, ²³¹Th, ²³²Th, ²³³Th, ²³⁴Th, ¹²³I, ¹²⁴I, ¹²⁵I, or ¹³¹I.

In any of the above methods or treatment protocols or regimens or kits,and unless otherwise specified, the compound including the radionuclidemay have a recognition moiety for a particular cancer, cancerous tissue,or other tissue-related material associated with a particular cancer.For example, where the cancer is prostate cancer, the compound mayinclude a recognition moiety for prostate specific membrane antigen,including for example, Glu-Urea based PSMA ligands. Such compoundsinclude those described in U.S. Pat. Nos. 8,211,401; 8,211,402;8,465,725; 8,487,129; and 8,562,945; and in PCT/US2014/011047. In someembodiments, the compound is MIP-1555, MIP-1519, MIP-1545, MIP-1427,MIP-1428, MIP-1379, MIP-1072, MIP-1095, MIP-1558, MIP-1405, or MIP-1404:

In the structural representations of MIP-1072 and MIP-1095, ^(x)I refersto a radionuclide of iodine. In the structural representations ofMIP-1558, MIP-1555, MIP-1545, MIP-1519, MIP-1427, MIP-1428, MIP-1405,MIP-1379, MIP-1405, and MIP-1404, M is a metal that is a radionuclide.In some embodiments, the compound is a Glu-urea-based PSMA ligand. Inany of the above methods or treatment protocols or regimens, unlessotherwise specified, the compound may be administered to the subjectfrom about 0.2 mg/kg to about 100 mg/kg. In some embodiments, the amountis from about 1 mg/kg to about 50 mg/kg. In yet other embodiments, theamount is from about 10 mg/kg to about 50 mg/kg. In some embodiments,the amount is from about 0.2 mg/kg to about 75 mg/kg. In someembodiments, the amount is from about 0.2 mg/kg to about 50 mg/kg. Insome embodiments, the amount is from about 0.2 mg/kg to about 25 mg/kg.In some embodiments, the amount is from about 0.2 mg/kg to about 10mg/kg. In some embodiments, the amount is from about 0.2 mg/kg to about5 mg/kg. In some embodiments, the amount is from about 1 mg/kg to about40 mg/kg. In some embodiments, the amount is from about 1 mg/kg to about30 mg/kg. In some embodiments, the amount is from about 1 mg/kg to about20 mg/kg. In some embodiments, the amount is from about 1 mg/kg to about10 mg/kg. In some embodiments, the amount is from about 10 mg/kg toabout 40 mg/kg. In some embodiments, the amount is from about 10 mg/kgto about 30 mg/kg. In some embodiments, the amount is from about 10mg/kg to about 20 mg/kg. In any of the above methods or treatmentprotocols or regimens, the compound may be administered to the subjectin an amount that is effective to displace the agent from the canceroustissue without substantially displacing the agent from non-canceroustissue.

In any of the above methods or treatment protocols or regimens or kits,and unless otherwise specified, the waiting period may be greater thanor equal to 1 hour, greater than or equal to 2 hours, greater than orequal to 4 hours, greater than or equal to 6 hours, greater than orequal to 8 hours, greater than or equal to 10 hours, greater than orequal to 12 hours, greater than or equal to 14 hours, greater than orequal to 16 hours, greater than or equal to 18 hours, greater than orequal to 20 hours, greater than or equal to 22 hours, or greater than orequal to 24 hours. In some embodiments, the waiting period is from about1 hour to about 60 hours. In some embodiments, the waiting period isfrom about 5 hours to about 36 hours. In some embodiments, the waitingperiod is about 24 hours. In some embodiments, the waiting period isabout 48 hours.

In any of the above methods or treatment protocols or regimens or kits,and unless otherwise specified, the agent is capable of displacing thecompound from non-cancerous tissue, while having a lesser effect ofdisplacing the compound from the cancerous tissue. In some embodiments,the agent does not include a radionuclide. In some embodiments, theagent is a compound of the same structure as the compound without theradionuclide. In other words, the agent may be cold version of thecompound. As used herein, a cold version of the compound is the compoundwithout a radionuclide. Where the compound includes a metal chelationmoiety, the cold version may be the free ligand, unchelated to a metal,or it may be chelated to a non-radioactive metal. Where the compoundincludes a radioactive non-metal, i.e., iodine, the cold version may bea non-radioactive iodine, or it may have a different group than iodine.In some embodiments, the recognition moiety of the agent includes aphosphinyl moiety.

The agent may be a more polar compound than the compound having theradionuclide.

In some embodiments, the agent has a recognition moiety for prostatespecific membrane antigen. In some embodiments, the agent includes aGlu-Urea-based PSMA ligand.

In some embodiments, the agent may be a cold MIP-1555, MIP-1072,MIP-1095, MIP-1558, or MIP-1404, or the agent may include, but is notlimited to, 2-[[methylhydroxyphosphinyl]methyl]pentanedioic acid;2-[[ethylhydroxyphosphinyl]methyl]pentanedioic acid;2-[[propylhydroxyphosphinyl]methyl]pentanedioic acid;2-[[butylhydroxyphosphinyl]methyl]pentanedioic acid;2-[[cyclohexylhydroxyphosphinyl]methyl]pentanedioic acid;2-[[phenylhydroxyphosphinyl]methyl]pentanedioic acid;2-[[(phenylmethyl)hydroxyphosphinyl]methyl]pentanedioic acid;2-[[((2-phenylethyl)methyl)hydroxyphosphinyl]methyl]pentanedioic acid;2-[[((3-phenylpropyl)methyl)hydroxyphosphinyl]methyl]pentanedioic acid;2-[[((3-phenylbutyl)methyl)hydroxyphosphinyl]methyl]pentanedioic acid;2-[[((2-phenylbutyl)methyl)hydroxyphosphinyl]methyl]pentanedioic acid;2-[[(4-phenylbutyl)hydroxyphosphinyl]methyl]pentanedioic acid;2-[[(aminomethyl)hydroxyphosphinyl]methyl]pentanedioic acid;7-(L-2-amino-2-carboxyethylthio)-2-(2,2-dimethylcyclopropanecarboxamide)-2-heptenoicacid; 2-(phosphonomethyl)pentanedioic acid;N-[methylhydroxyphosphinyl]glutamic acid;N-[ethylhydroxyphosphinyl]glutamic acid;N-[propylhydroxyphosphinyl]glutamic acid;N-[butylhydroxyphosphinyl]glutamic acid;N-[phenylhydroxyphosphinyl]glutamic acid;N-[(phenylmethyl)hydroxyphosphinyl]glutamic acid;N-[((2-phenylethyl)methyl)hydroxyphosphinyl]glutamic acid;N-methyl-N-[phenylhydroxyphosphinyl]glutamic acid;2-(3-mercaptopropyl)pentanedioic acid (2-MPPA), a pharmaceuticallyacceptable salt thereof; or a mixture of any two or more thereof. Insome embodiments, the agent includes 2-(phosphonomethyl)pentanedioicacid. In some embodiments, the agent includes cold MIP-1555, coldMIP-1519, cold MIP-1545, cold MIP-1427, cold MIP-1428, cold MIP-1379,cold MIP-1072, cold MIP-1095, cold MIP-1558, cold MIP-1405, or coldMIP-1404.

The agent may be administered to the subject in an amount that iseffective to displace the compound from the non-cancerous tissue withoutsubstantially displacing the compound from cancerous tissue. The amountof the agent administered will depend upon the particular compound andradionuclide that are administered, and the type of agent that isadministered. For example, but not by way of limitation, the amount ofagent administered may be from about 0.2 mg/kg to about 100 mg/kg. Insome embodiments the agent is administered from about 1 mg/kg to about50 mg/kg. In yet other embodiments, the agent is administered to thesubject from about 10 mg/kg to about 50 mg/kg. In some embodiments, theagent is administered to the subject from about 0.2 mg/kg to about 75mg/kg. In some embodiments, the agent is administered to the subjectfrom about 0.2 mg/kg to about 50 mg/kg. In some embodiments, the agentis administered to the subject from about 0.2 mg/kg to about 25 mg/kg.In some embodiments, the agent is administered to the subject from about0.2 mg/kg to about 10 mg/kg. In some embodiments, the agent isadministered to the subject from about 0.2 mg/kg to about 5 mg/kg. Insome embodiments, the agent is administered to the subject from about 1mg/kg to about 40 mg/kg. In some embodiments, the agent is administeredto the subject from about 1 mg/kg to about 30 mg/kg. In someembodiments, the agent is administered to the subject from about 1 mg/kgto about 20 mg/kg. In some embodiments, the agent is administered to thesubject from about 1 mg/kg to about 10 mg/kg. In some embodiments, theagent is administered to the subject from about 10 mg/kg to about 40mg/kg. In some embodiments, the agent is administered to the subjectfrom about 10 mg/kg to about 30 mg/kg. In some embodiments, the agent isadministered to the subject from about 10 mg/kg to about 20 mg/kg.

The present invention, thus generally described, will be understood morereadily by reference to the following examples, which are provided byway of illustration and are not intended to be limiting of the presentinvention.

EXAMPLES

Material and Methods. All animal studies were approved by theInstitutional Animal Care and Use Committee in accordance with theguidelines for Care and Use of Laboratory Animals.

With regard to Examples 1-3, mice were housed under standard conditionsin approved facilities with 12 hour light/dark cycles and given food andwater ad libidum. BALB/c nude mice were implanted with 5×10⁶ LNCaP cells(BD Biosciences) suspended in Matrigel (BD Biosciences) behind the leftshoulder. The mice were used for our study after 8-12 weeks, when thetumors reached approximately 1.5 cm in diameter.

MIP-1095, MIP-1404, and MIP-1558. The synthesis of MIP-1095(S)-2-(3-((S)-1-carboxy-5-(3-(4-iodophenyl)ureido)pentyl)ureido)pentanedioicacid, the radiolabeling precursor trimethyltin-MIP-1095 and thesubsequent radiolabeling with a variety of iodine isotopes was describedpreviously.^(6,8) An illustrative structural representation of MIP-1095is shown below as the ¹²³I compound; however, as eluded to previously,other iodine isotopes such as ¹²⁴I, ¹²⁵I, and ¹²³I may be used.

MIP-1404 and MIP-1558 were provided from Molecular InsightPharmaceuticals (Tarrytown, N.Y., USA) and labeled with either ^(99m)Tc(ELUMATIC III generator, IBA, Belgium) or ⁶⁸Ga (Ga-Generator, iThembaLABS, Somerset West, South Africa). The synthesis of MIP-1404 hasalready been described previously.⁹ The structure of MIP-1404 isillustrated below as a metal complexed “M” compound. The synthesis ofMIP-1558 has already been previously described in WO 2014/110372. Thestructure of MIP-1558 is illustrated below as a metal complexed “M”compound.

Example 1. Mice bearing LNCaP tumors of 1.5 cm diameter were injectedvia the tail vein with 37 MBq of ¹²⁵I-MIP-1095 in 100 μl at a specificactivity of >1,000 mCi/Amol. Mice were imaged at a baseline time of 16hours post injection giving enough time for tracer clearance from theblood stream and kidney calices. Immediately after the baseline scan,PMPA in 100 μl physiological saline solution was injected via the tailvein in doses of 50 mg/kg, 10 mg/kg, 1 mg/kg, and 0.2 mg/kg (n=3,respectively), or the animals served as controls (n=5). Planar scanswere obtained at 2 hours, 4 hours, 6 hours, and 24 hours (1 day) postPMPA injection. See FIG. 1A.

Example 2. MIP-1404 was evaluated after labeling with ^(99m)Tc in LNCaPbearing BALB/c nude mice. The baseline scan was done 1 hour afterinjection of 9.3 to 11 MBq ^(99m)Tc-MIP-1404 in 100 μl directly followedwith the injection of 50 mg/kg PMPA in 50 μl or 50 μl saline (n=3,respectively). Planar scans were obtained 1 hour and 3 hours after thesubsequent PMPA/saline injection. The flow chart of the experiment ispresented in FIG. 3A. Quantification was done with kidney regions ofinterest (ROI) arising counts per minute (cpm) which were then convertedto percentage-of-baseline values. The same protocol was used to evaluatekidney uptake of 10.3 to 11.6 MBq ^(99m)Tc-MIP-1404 in 100 μl directlyfollowed with the injection of 50 mg/kg, 10 mg/kg, and 1 mg/kg PMPA in50 μl or saline as controls (n=4, respectively) in non-tumor bearingNMRI mice. In a pilot experiment planar scans after injection of thesalivary gland tracer ^(99m)Tc pertechnetate were acquired and thediagnostic value of the images was judged visually.

Example 3. MIP-1558 was evaluated after labeling with ⁶⁸Ga and imagingin an animal PET. Due to the short half-life of ⁶⁸Ga (68 min) thebaseline scan was done 1 hour after injection of 9 to 28 MBq⁶⁸Ga-MIP-1558 in 100 μl into LNCaP tumor bearing BALB/c nude micedirectly followed with the injection of 50 mg/kg PMPA (n=3) in 50 μl oran equivocal volume of saline as controls (n=3). Post-PMPA scans wereacquired 1 hour and 2 hours after administration of PMPA. Both tumor andkidney uptake was determined in a volume of interest (VOI). Serial⁶⁸Ga-MIP-1558 PET scans were also done with three rats, once with salineand a few days later with serial co-injection of PMPA (10 mg/kg), fordelineation of the salivary glands in larger animals.

Imaging. During imaging, mice were anesthetized using 1% isofluorane gasin oxygen flowing at 0.6 L/min. Serial planar scans were done with aGamma Imager-sct (biospace labs, Paris, France). Quantification of tumorand kidney uptake was done by determining the counts per minute (cpm) inthe target region by ROI-techniques. The baseline scan beforeadministration of PMPA served as reference, the succeeding images arereported in percentage of the baseline value. PET scans were done with adedicated small animal PET scanner (Siemens, Iveon) and quantificationwas done by VOI-techniques and reported as standardized uptake values(SUV).

Results. MIP1095: Subsequent injection of PMPA 16 hours after MIP-1095translated into a rapid and quantitative relevant displacement of renalactivity with all PMPA doses. Scintigraphy scans from one example out ofeach group are presented in FIG. 1B. The course of residual activity of¹²⁵I-MIP-1095 measured at the particular time points after subsequentadministration of 0.2 mg/kg, 1 mg/kg, 10 mg/kg, and 50 mg/kg PMPA, orcontrols, are presented in FIG. 2A for kidney and FIG. 2B for tumorROIs. Even at a very low dose of 0.2 mg/kg, there is a high significant(p<0.01) displacement from the kidneys. The effect was more pronouncedat higher doses and showed a dose dependency with 1 mg/kg, thusrevealing an even higher kidney washout than the 0.2 mg/kg dose(p<0.01). However, doses greater than 1 mg/kg did not improve thekidney-protective effect (i.e., the differences between 1 mg/kg, 10mg/kg, and 50 mg/kg PMPA groups were not significant). In tumor tissue,the uptake of ¹²⁵I-MIP-1095 was displaced by subsequent injection ofPMPA. By visual observation, the effect appears to be dose dependent:residual uptake 2 hours after PMPA was 98% with 0.2 mg/kg, 93% with 1mg/kg, 77% with 10 mg/kg, and 75% with 50 mg/kg. However, due to thesmall size of the group (n=3) none of the differences was statisticallysignificant.

MIP-1404. Normalized to the 1 hour post injection tumor uptake there isa fast wash-out of MIP-1404 from the kidneys even without intervention.When 50 mg/kg PMPA was administered directly following the baselinescan, the washout from kidney was significantly (p<0.01) enhanced at 2and 4 hours (FIG. 3B and FIG. 4A). In comparison to the controls, thedifference in tumor uptake measured in the PMPA group was notsignificant, with the 50 mg/kg PMPA dose (FIG. 4B). As MIP-1404 seemedrather robust against tumor displacement even after high dose PMPA thedilution series with 10 mg/kg and 1 mg/kg were done in non-tumor bearingNMRI mice. Residual kidney uptake at 2 and 4 hours were 36% and 15%,respectively, in controls, 13% and 9%, respectively, after 1 mg/kg PMPA,15 and 10%, respectively, after 10 mg/kg PMPA, and 11% and 7%,respectively, after 50 mg/kg PMPA. This pattern is in concert with thelarger MIP-1095 series, with optimal kidney displacement even at 1 mg/kgand no benefit of higher PMPA doses. In a pilot study, planar scans ofthe mice were acquired after injection of ^(99m)Tc pertechnetate whichis known to accumulate in the salivary glands but due to the limitedspatial resolution of the camera these small structures were not able tobe sufficiently delineated in the mice.

MIP-1558: In controls, the mean tumor uptake of ⁶⁸Ga-MIP1558 was 1 hourpost injection SUVmax 0.95, 2 hours post injection SUVmax 1.25, and 3hours post injection SUVmax 1.24. The 1 hour post injection baselinescan of the PMPA group was quite similar with a mean tumor uptake withan SUVmax of 0.94. Following administration of 50 mg/kg PMPA, the 2hours post injection SUVmax decreased to a mean of 0.40, and the 3 hourspost injection SUVmax decreased to 0.39. The corresponding kidney uptakein controls vs. PMPA at 1 hour, 2 hours, and 3 hours post injection wereSUVmax values of 5.83 vs 7.13, 3.13 vs 1.87, and 2.60 vs 1.77,respectively. When examining the salivary region in the rats, aspontaneous SUVmax of mean 0.95 at 1 hour post injection, and 0.24 at 3hours post injection was observed, thus, the perfusion dependency seemslarge in comparison to the specific long term binding. In the PMPAexperiment an SUVmax of mean 0.82 at 1 hour post injection and 0.23 at 3hours post injection were observed, thus, there does not appear to be ameasurable change due to the PMPA intervention. However, on the latertime points the salivary glands were hardly delineable compared to thebackground noise. It is believed that in larger animals and humans, thatsalivary gland, lacrimal gland, and parotid gland differentiation willbe observable.

Discussion. The data demonstrate that injection of PMPA following aninduction period after radionuclide therapy administration, provides forradiation dose limiting kidney uptake, and, possibly radiation doselimiting uptake by the salivary, lacrimal, and parotid glands. Arelevant kidney displacement was observed independent of whether theevaluated tracer included a chelate (MIP-1404, MIP-1558) or not(MIP-1095). In addition to some nonspecific kidney uptake due to tubularreabsorption which might be further improved by modifications affectingthe chelate or linker of the tracer molecule, a relevant part of kidneyuptake seems to be related to specific PSMA binding.

PSMA is an emerging target for imaging and radionuclide therapy ofmetastasized PCa. Radionuclide therapy compounds such as, but notlimited to, MIP-1095, MIP-1404 and MIP-1558, are currently among themost promising PSMA-ligands. The promise is attributed to: (a) fasttumor targeting, (b) fast clearance from untargeted organs, and (c)sufficient residency times in tumor tissue due to ligand-inducedcellular internalization. MIP-1404 is of particular interest because itis a single amino acid chelator (SAAC) that can be either labeled withdiagnostic ^(99m)Tc or in therapeutic intention with ¹⁸⁶Re or ¹⁸⁸Re.Technetium and rhenium are chemically related and share structural aswell as reactive similarities. ^(99m)Tc is available from approvedgenerator systems and can be imaged with the numerous already installedAnger cameras (i.e., gamma camera or scintillation camera). ¹⁸⁶Re(half-life 3.7 days, max. energy of beta-emission 1.07 MeV, 11%co-emission of 137 keV gammas for imaging) presents an attractive“matched pair” for therapy. ¹⁸⁸Re (half-life 17 h, max. energy ofbeta-emission 2.12 MeV, 15% co-emission of 155 keV gammas for imaging)can be obtained from a ¹⁸⁸W/¹⁸⁸Re generator system (Oak Ridge NationalLaboratory) which was FDA approved and would be suitable for clinicalapplication. MIP-1558 is clinically interesting because its DOTA(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) chelate canbe labeled with the generator product ⁶⁸Ga for PET-imaging or ¹¹¹In forpre-therapeutic dosimetry studies. Numerous beta and alpha particleemitters—e.g. ⁹⁰Y, ¹⁷⁷Lu, ₂₁₃Bi, ²²⁵Ac—are promising “matched pairs” fortherapy [KRAC CCR2011, EJNM2014]. MIP-1095 can be tagged with differentisotopes of iodine. Labeled with ¹²⁵I (half-life 60 days, gamma emission27-35 keV), MIP-1095 presents the best characteristics for small animalimaging, and, therefore, it was chosen for the main experiment.

It is common practice to show specific binding of PSMA ligands bysimultaneous coinjection of 50 mg/kg PMPA which results in a completeblocking of MIP-1095 binding sites in tumor and kidneys.^(3,11) Ourfindings now implicate, that a subsequent injection of the competitor,or blocking agent, does not significantly displace the endosome-fixedligand from tumor, while displacing the non-internalized ligands fromthe kidneys and the salivary, lacrimal, and parotid glands.

The salivary glands present another organ with a high uptake ofdiagnostic PSMA-tracers and also to ¹³¹I-MIP-1095. In the present smallanimal imaging experiments, sufficient delineation of the salivaryglands was not possible by scintigraphy or even dedicated small animalPET of rats. Therefore, the situation in salivary glands is ratherunclear. However, if PSMA expression is responsible for traceraccumulation in the salivary glands, PMPA, or other agents/blockers mayalso be suitable to displace radioactive PSMA ligands from these organsand, as such, treat xerostomia side effects of PSMA targetedradionuclide therapy.

With regard to kidneys, a dose of PMPA in the range of 0.2 to 1.0 mg/kgwas used. A dose of 1.0 mg/kg translates into near total renaldisplacement with only minimal (<10%) effect on tumor uptake. With 0.2mg/kg the decrease in tumor uptake was <5%, but still a relevantimprovement of kidney uptake could be observed. Nevertheless, renalactivity was only sub-totally displaced with the lower dose. Doing aconservative, body surface based, extrapolation from the 0.2 mg/kg mousedose to men, an 80 kg patient should receive about 60 mg of PMPA.

The orally bioavailable PSMA-inhibitor 2-(3-mercaptopropyl)pentanedioicacid (2-MPPA) which is nearly as potent as2-(phosphonomethyl)pentanedioic acid (2-PMPA)—IC₅₀ 85 nM vs. 30 nM¹²—wasalready evaluated in 25 healthy subjects with a mean body weight of 71kg. Doses of up to 750 mg (i.e., ˜10 mg/kg) were found safe andgenerally well tolerated.

Example 4. The effect of cold MIP-1095 on the distribution of¹²⁵I-MIP-1095 in subcutaneously-implanted LNCaP xenografts in maleTaconic Nude mice (CrTac:NCr-Foxn1^(nu)) using an automatedgamma-counter was evaluated. Blood and tissues were collected 18 hoursafter intravenous injection of ¹²⁵I-MIP-1095 (2 hours after displacingagent injections). Gamma-counter readouts were obtained for each sampleand expressed as percent injected dose per gram of tissue usinggamma-counter readout of the dosing solution as a reference.

PMPA was formulated in a vehicle of sterile saline. For dosing of the0.50 mg/kg group, the PMPA was placed into a sterile vial and theappropriate amount of saline was added and mixed well by vortexing toform a clear solution with a pH value of 3.56. The dosing solution wasprepared fresh the day of administration.

Cold MIP-1095 was stored protected from light at −80° C. until use. Thecompound was formulated in a vehicle of sterile saline. For dosing ofthe 0.50 mg/kg group, the cold MIP-1095 was placed into a sterile vialand the appropriate amount of saline was added and mixed well byvortexing to form a clear solution with a pH value of 6.96. Dosingsolutions for the 0.15 mg/kg and 0.05 mg/kg groups were prepared bydirect (not serial) dilution of the dosing solution from the 0.5 mg/kggroup with complete vehicle. The dosing solutions were prepared freshthe day of administration.

¹²⁵I-MIP-1095 was frozen on dry ice with an activity concentration of0.2 mCi/ml, and stored at −80° C. until use. For dosing, the stocksolution (colorless) was thawed in a 37° C. water bath and diluted 1:1with sterile saline for a fixed 0.1 mCi/ml solution for dosing.Additionally, it was spiked with cold MIP-1095 to a final concentration0.00125 mg/mL. This approximately corresponded to a chemical massassociated with the therapeutically-active dose of ¹³¹I-MIP-1095 (bodysurface area-corrected).

LNCaP (clone FGC) cells were obtained from ATCC. They were grown in RPMI1640 medium which was modified with 1% 100 mM Na pyruvate, 1% 1M HEPESbuffer, 2.5 g/L Glucose and supplemented with 10% non-heat-inactivatedFetal Bovine Serum (FBS) and 1% 100× Penicillin/Streptomycin/L-Glutamine(PSG). The growth environment was maintained in an incubator with a 5%CO₂ atmosphere at 37° C. When expansion was complete, the cells (passage4) were trypsinized using 0.25% trypsin-EDTA solution. Following celldetachment, the trypsin was inactivated by dilution with complete growthmedium and any clumps of cells were separated by pipetting. The cellswere centrifuged at 200 rcf for 10 minutes at 4° C., the supernatant wasaspirated, and the pellet was re-suspended in cold Dulbecco's PhosphateBuffered Saline (DPBS) by pipetting. An aliquot of the homogeneous cellsuspension was diluted in a trypan blue solution and counted using aLuna automated cell counter. The pre-implantation cell viability was97%. The cell suspension was centrifuged at 200 rcf for 10 minutes at 4°C. The supernatant was aspirated and the cell pellet was re-suspended incold 50% serum-free medium: 50% Matrigel® to generate a finalconcentration of 2.50E+07 trypan-excluding cells/ml. The cell suspensionwas maintained on wet ice during implantation. Following implantation,an aliquot of the remaining cells was diluted with a trypan bluesolution and counted to determine the post-implantation cell viability(96%).

Test animals were implanted subcutaneously, high in the axilla (justunder the fore limb) on Day 0 with 5.0E+06 cells in 0.2 ml of 50%serum-free medium: 50% Matrigel® using a 27-gauge needle and syringe.

All mice were sorted into study groups based on caliper measurementestimation of tumor burden. The mice were distributed to ensure that themean tumor burden for all groups was within 10% of the overall meantumor burden for the study population. Treatment began on Day 25. Micewere administered test agent via intravenous injections. The mice wereadministered a fixed volume injection of 100 μl (approximately 4 ml/kg)via the tail vein, using a 28-gauge needle and insulin syringe. A freshneedle and syringe was used for each mouse.

Group 1—¹²⁵I-MIP-1095+Saline, 0.4 mCi/kg (0.005 mg/kg)+100 μl fixedvolume, IV+IV, “Control”; test article at 0 hours, saline 16 hours posttest article.

Group 2—¹²⁵I-MIP-1095+PMPA, 0.4 mCi/kg (0.005 mg/kg)+0.5 mg/kg, IV+IV,“Positive control”; test article at 0 hours, PMPA 16 hours post testarticle.

Group 3—¹²⁵I-MIP-1095+Cold MIP-1095 (conc.1), 0.4 mCi/kg (0.005mg/kg)+0.05 mg/kg, IV+IV, test article at 0 hours, cold MIP-1095(conc. 1) 16 hours post test article.

Group 4—¹²⁵I-MIP-1095+Cold MIP-1095 (conc.2), 0.4 mCi/kg (0.005mg/kg)+0.15 mg/kg, IV+IV, test article at 0 hours, cold MIP-1095 (conc.2) 16 hours post test article.

Group 5—¹²⁵I-MIP-1095+Cold MIP-1095 (conc.3), 0.4 mCi/kg (0.005mg/kg)+0.5 mg/kg, IV+IV, test article at 0 hours, cold MIP-1095 (conc.3) 16 hours post test article.

At 18 hours after the ¹²⁵I-MIP-1095 dose, 5 mice per group, in numericalorder, were euthanized for blood and tissue collection. Dosing timeswere staggered to allow for sample collection. All mice were euthanizedvia over exposure to carbon dioxide. Whole blood was collected viacardiac puncture and placed to a cylindrical test tube for gammacounting. After whole blood collection, the following tissues wereexcised: heart, lungs, liver, spleen, salivary glands, kidneys, stomach(with contents), large intestine (with contents), small intestines (withcontents), testes, quadriceps (from right hind limb), femur (from righthind limb), brain, trachea/thyroid, adipose, and tumor. Each tissue wasplaced into a tared, cylindrical test tube and tissue wet weights wererecorded. All mice were necropsied as they exited the study.

The samples were then transported to the gamma counter, where sampleswere counted for sixty seconds using a PerkinElmer WIZARD² gammacounting system. A 100 μl standard aliquot (1 dose) of the ¹²⁵I-MIP-1095dosing solution was also measured along with the tissues. This aliquotwas intended to represent the total injected dose for mice to enable asimplified calculation of percent injected dose (% ID). The % ID wascalculated as the ratio of the total individual tissue counts to thetotal counts acquired from the standard aliquot×100. % ID/g wascalculated by dividing the % ID by the tissue wet weight in grams, asmeasured at the time of sampling. There was a time delay between samplecollections and gamma counting, the measured activity values werecorrected for decay as needed. See FIGS. 5 and 6.

Results/Discussion. In the negative control group (group 1; saline),LNCaP tumors showed the highest exposure of ¹²⁵I-MIP-1095 followed bykidneys, stomach, small and large intestines, and liver. All othertissues and organs (including salivary glands) showed the exposure lessthan 1% of ID/g. It appears that mouse is a suitable model to evaluatethe displacing effect in the kidneys but not a suitable model forsalivary glands which is another potential organ for radiation toxicityassociated with ¹³¹I-MIP-1095 therapy (see Eur. J. Nucl. Med. Mol.Imaging. 2014 July; 41(7): 1280-92).

In the positive control group (group 2; 0.5 mg/kg PMPA), the displacingagent greatly reduced exposure of ¹²⁵I-MIP-1095 in the kidneys while theexposure in LNCaP tumors and all other organs and tissues was littlechanged. These results are in good correlation with theliterature-reported data (see J. Nucl. Med. 2015 February; 56(2):293-8).

In the test article groups (groups 3, 4 and 5; cold MIP-1095 at 0.05,0.15 and 0.5 mg/kg, respectively), the displacing agent dose-dependentlyreduced ¹²⁵I-MIP-1095 kidney exposure. At the highest dose, thereduction in kidney exposure was equivalent to the one observed forpositive control. The reduction in kidney exposure, however, wasaccompanied by a dose-dependent decrease in LNCaP tumors exposure and adose-dependent increase in liver, stomach, large and small intestinesexposure. The changes in tumor and normal organs exposure were not aspronounced as changes in kidney exposure. For example, in group 4, thekidney exposure was reduced 5-fold compared to negative control whileLNCaP tumors exposure was reduced only 1.2-fold while exposure in otherorgans was little changed.

Overall, the study results indicated that with careful dose selection itis possible to meaningfully reduce kidney exposure by administering acold MIP-1095 several hours post ¹²⁵I-MIP-1095 dose while maintaining areasonably high tumor exposure and keeping all other organs exposurerelatively unchanged.

REFERENCES

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EQUIVALENTS

While certain embodiments have been illustrated and described, it shouldbe understood that changes and modifications can be made therein inaccordance with ordinary skill in the art without departing from thetechnology in its broader aspects as defined in the following claims.

The embodiments, illustratively described herein may suitably bepracticed in the absence of any element or elements, limitation orlimitations, not specifically disclosed herein. Thus, for example, theterms “comprising,” “including,” “containing,” etc. shall be readexpansively and without limitation. Additionally, the terms andexpressions employed herein have been used as terms of description andnot of limitation, and there is no intention in the use of such termsand expressions of excluding any equivalents of the features shown anddescribed or portions thereof, but it is recognized that variousmodifications are possible within the scope of the claimed technology.Additionally, the phrase “consisting essentially of” will be understoodto include those elements specifically recited and those additionalelements that do not materially affect the basic and novelcharacteristics of the claimed technology. The phrase “consisting of”excludes any element not specified.

The present disclosure is not to be limited in terms of the particularembodiments described in this application. Many modifications andvariations can be made without departing from its spirit and scope, aswill be apparent to those skilled in the art. Functionally equivalentmethods and compositions within the scope of the disclosure, in additionto those enumerated herein, will be apparent to those skilled in the artfrom the foregoing descriptions. Such modifications and variations areintended to fall within the scope of the appended claims. The presentdisclosure is to be limited only by the terms of the appended claims,along with the full scope of equivalents to which such claims areentitled. It is to be understood that this disclosure is not limited toparticular methods, reagents, compounds compositions or biologicalsystems, which can of course vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to be limiting.

In addition, where features or aspects of the disclosure are describedin terms of Markush groups, those skilled in the art will recognize thatthe disclosure is also thereby described in terms of any individualmember or subgroup of members of the Markush group.

As will be understood by one skilled in the art, for any and allpurposes, particularly in terms of providing a written description, allranges disclosed herein also encompass any and all possible subrangesand combinations of subranges thereof. Any listed range can be easilyrecognized as sufficiently describing and enabling the same range beingbroken down into at least equal halves, thirds, quarters, fifths,tenths, etc. As a non-limiting example, each range discussed herein canbe readily broken down into a lower third, middle third and upper third,etc. As will also be understood by one skilled in the art all languagesuch as “up to,” “at least,” “greater than,” “less than,” and the like,include the number recited and refer to ranges which can be subsequentlybroken down into subranges as discussed above. Finally, as will beunderstood by one skilled in the art, a range includes each individualmember.

All publications, patent applications, issued patents, and otherdocuments referred to in this specification are herein incorporated byreference as if each individual publication, patent application, issuedpatent, or other document was specifically and individually indicated tobe incorporated by reference in its entirety. Definitions that arecontained in text incorporated by reference are excluded to the extentthat they contradict definitions in this disclosure.

Other embodiments are set forth in the following claims.

What is claimed is:
 1. A therapeutic radionuclide regimen for treatingcancerous prostate tissue in a subject, the regimen comprisingadministering an agent to the subject and after allowing a waitingperiod of 1 hour to 60 hours to pass administering a compound comprisinga recognition moiety for Prostate Specific Membrane Antigen (“PSMA”) anda radionuclide to a subject harboring the cancerous prostate tissue;wherein: the compound binds to both cancerous prostate tissue andnon-cancerous tissue; the compound is administered in an amountsufficient to cause a displacement of the agent in cancerous tissue; theagent does not include a radionuclide; the compound is a Glu-urea-basedPSMA ligand; and the agent comprises a Glu-urea-based PSMA ligand, aphosphinyl containing moiety,7-(L-2-amino-2-carboxyethylthio)-2-(2,2-dimethylcyclopropanecarboxamide)-2-heptenoicacid, 2-(phosphonomethyl)pentanedioic acid, or2-(3-mercaptopropyl)pentanedioic acid (2-MPPA).
 2. The regimen of claim1, wherein the non-cancerous tissue is kidney tissue, salivary glandtissue, lacrimal gland tissue, parotid gland tissue, or small intestinetissue.
 3. The regimen of claim 1, wherein the radionuclide is aradioactive isotope of Ga, I, Y, Lu, Bi, Ac, Re, In, Th, or Tc.
 4. Theregimen of claim 1, wherein the compound is:

and wherein M is a radionuclide and ^(x)I is a radionuclide of iodine.5. The regimen of claim 1, wherein the agent comprises:2-[[methylhydroxyphosphinyl]methyl]pentanedioic acid;2-[[ethylhydroxyphosphinyl]methyl]pentanedioic acid;2-[[propylhydroxyphosphinyl]methyl]pentanedioic acid;2-[[butylhydroxyphosphinyl]methyl]pentanedioic acid;2-[[cyclohexylhydroxyphosphinyl]methyl]pentanedioic acid;2-[[phenylhydroxyphosphinyl]methyl]pentanedioic acid;2-[[(phenylmethyl)hydroxyphosphinyl]methyl]pentanedioic acid;2-[[((2-phenylethyl)methyl)hydroxyphosphinyl]methyl]pentanedioic acid;2-[[((3-phenylpropyl)methyl)hydroxyphosphinyl]methyl]pentanedioic acid;2-[[((3-phenylbutyl)methyl)hydroxyphosphinyl]methyl]pentanedioic acid;2-[[((2-phenylbutyl)methyl)hydroxyphosphinyl]methyl]pentanedioic acid;2-[[(4-phenylbutyl)hydroxyphosphinyl]methyl]pentanedioic acid;2-[[(aminomethyl)hydroxyphosphinyl]methyl]pentanedioic acid;7-(L-2-amino-2-carboxyethylthio)-2-(2,2-dimethylcyclopropanecarboxamide)-2-heptenoicacid; 2-(phosphonomethyl)pentanedioic acid;N-[methylhydroxyphosphinyl]glutamic acid;N-[ethylhydroxyphosphinyl]glutamic acid;N-[propylhydroxyphosphinyl]glutamic acid;N-[butylhydroxyphosphinyl]glutamic acid;N-[phenylhydroxyphosphinyl]glutamic acid;N-[(phenylmethyl)hydroxyphosphinyl]glutamic acid;N-[((2-phenylethyl)methyl)hydroxyphosphinyl]glutamic acid;N-methyl-N-[phenylhydroxyphosphinyl]glutamic acid;2-(3-mercaptopropyl)pentanedioic acid (2-MPPA), a pharmaceuticallyacceptable salt thereof; or a mixture of any two or more thereof.
 6. Theregimen of claim 1, wherein the agent is cold MIP-1555, cold MIP-1519,cold MIP-1545, cold MIP-1427, cold MIP-1428, cold MIP-1379, coldMIP-1072, cold MIP-1095, cold MIP-1558, cold MIP-1405, or cold MIP-1404;wherein:

and M is absent or is a non-radionuclide metal and ^(x)I is anon-radionuclide of iodine.
 7. The regimen of claim 1, wherein thecompound is administered to the subject from about 0.2 mg/kg to about100 mg/kg and the agent is administered to the subject from about 0.2mg/kg to about 100 mg/kg.
 8. A treatment protocol for a subjectdiagnosed with cancer, the protocol comprising: a) administering anagent to the subject; b) after allowing a waiting period of 1 hour to 60hours to pass; and then c) administering a compound comprising arecognition moiety for Prostate Specific Membrane Antigen (“PSMA”) and aradionuclide to the subject; wherein: the compound binds to bothcancerous prostate tissue and non-cancerous tissue; the compound isadministered in an amount sufficient to cause a displacement of theagent in cancerous tissue; the agent does not include a radionuclide;the compound is a Glu-urea-based PSMA ligand; and the agent comprises aGlu-urea-based PSMA ligand, a phosphinyl containing moiety,7-(L-2-amino-2-carboxyethylthio)-2-(2,2-dimethylcyclopropanecarboxamide)-2-heptenoic acid,2-(phosphonomethyl)pentanedioic acid, or2-(3-mercaptopropyl)pentanedioic acid (2-MPPA).
 9. The treatmentprotocol of claim 8, wherein at least one of steps a) and c) arerepeated at periodic intervals.
 10. The treatment protocol of claim 8,wherein steps a), b), and c) are repeated.
 11. The treatment protocol ofclaim 8 further comprising d) monitoring treatment by imaging usingscintigraphy, single-photon emission computed tomography (SPECT), orpositron emission tomography (PET).
 12. The treatment protocol of claim8, wherein the non-cancerous tissue is kidney tissue, salivary glandtissue, lacrimal gland tissue, parotid gland tissue, or small intestinetissue.
 13. The treatment protocol of claim 8, wherein the radionuclideis a radioactive isotope of Ga, I, Y, Lu, Bi, Ac, Re, In, Th, or Tc. 14.The treatment protocol of claim 8, wherein the compound is:

and wherein M is a radionuclide and ^(x)I is a radionuclide of iodine.15. The treatment protocol of claim 8, wherein the agent comprises:2-[[methylhydroxyphosphinyl]methyl]pentanedioic acid;2-[[ethylhydroxyphosphinyl]methyl]pentanedioic acid;2-[[propylhydroxyphosphinyl]methyl]pentanedioic acid;2-[[butylhydroxyphosphinyl]methyl]pentanedioic acid;2-[[cyclohexylhydroxyphosphinyl]methyl]pentanedioic acid;2-[[phenylhydroxyphosphinyl]methyl]pentanedioic acid;2-[[(phenylmethyl)hydroxyphosphinyl]methyl]pentanedioic acid;2-[[((2-phenylethyl)methyl)hydroxyphosphinyl]methyl]pentanedioic acid;2-[[((3-phenylpropyl)methyl)hydroxyphosphinyl]methyl]pentanedioic acid;2-[[((3-phenylbutyl)methyl)hydroxyphosphinyl]methyl]pentanedioic acid;2-[[((2-phenylbutyl)methyl)hydroxyphosphinyl]methyl]pentanedioic acid;2-[[(4-phenylbutyl)hydroxyphosphinyl]methyl]pentanedioic acid;2-[[(aminomethyl)hydroxyphosphinyl]methyl]pentanedioic acid;7-(L-2-amino-2-carboxyethylthio)-2-(2,2-dimethylcyclopropanecarboxamide)-2-heptenoicacid; 2-(phosphonomethyl)pentanedioic acid;N-[methylhydroxyphosphinyl]glutamic acid;N-[ethylhydroxyphosphinyl]glutamic acid;N-[propylhydroxyphosphinyl]glutamic acid;N-[butylhydroxyphosphinyl]glutamic acid;N-[phenylhydroxyphosphinyl]glutamic acid;N-[(phenylmethyl)hydroxyphosphinyl]glutamic acid;N-[((2-phenylethyl)methyl)hydroxyphosphinyl]glutamic acid;N-methyl-N-[phenylhydroxyphosphinyl]glutamic acid;2-(3-mercaptopropyl)pentanedioic acid (2-MPPA), a pharmaceuticallyacceptable salt thereof; or a mixture of any two or more thereof. 16.The treatment protocol of claim 8, wherein the agent is a cold form ofany one or more of:

and M is absent or is a non-radionuclide metal and ^(x)I is anon-radionuclide of iodine.
 17. The treatment protocol of claim 8,wherein the compound is administered to the subject from about 0.2 mg/kgto about 100 mg/kg, and the agent is administered to the subject fromabout 0.2 mg/kg to about 100 mg/kg.